1. Field of the Invention
This invention relates to the field of bioscience in which arrays of oligonucleotide probes, fabricated or deposited on a surface, are used to identify DNA sequences in cell matter. The present invention has a wide range of application for synthesis of arrays for conducting cell study, for diagnosing disease, identifying gene expression, monitoring drug response, determination of viral load, identifying genetic polymorphisms, and the like.
Significant morbidity and mortality are associated with infectious diseases and genetically inherited disorders. More rapid and accurate diagnostic methods are required for better monitoring and treatment of these conditions. Molecular methods using DNA probes, nucleic acid hybridization and in vitro amplification techniques are promising methods offering advantages to conventional methods used for patient diagnoses.
Nucleic acid hybridization has been employed for investigating the identity and establishing the presence of nucleic acids. Hybridization is based on complementary base pairing. When complementary single stranded nucleic acids are incubated together, the complementary base sequences pair to form double-stranded hybrid molecules. The ability of single stranded deoxyribonucleic acid (ssDNA) or ribonucleic acid (RNA) to form a hydrogen bonded structure with a complementary nucleic acid sequence has been employed as an analytical tool in molecular biology research. The availability of radioactive nucleoside triphosphates of high specific activity and the development of methods for their incorporation into DNA and RNA has made it possible to identify, isolate, and characterize various nucleic acid sequences of biological interest. Nucleic acid hybridization has great potential in diagnosing disease states associated with unique nucleic acid sequences. These unique nucleic acid sequences may result from genetic or environmental change in DNA by insertions, deletions, point mutations, or by acquiring foreign DNA or RNA by means of infection by bacteria, molds, fumgi, and viruses.
The application of nucleic acid hybridization as a diagnostic tool in clinical medicine is limited due to the cost and effort associated with the development of sufficiently sensitive and specific methods for detecting potentially low concentrations of disease-related DNA or RNA present in the complex mixture of nucleic acid sequences found in patient samples.
One method for detecting specific nucleic acid sequences generally involves immobilization of the target nucleic acid on a solid support such as nitrocellulose paper, cellulose paper, diazotized paper, or a nylon membrane. After the target nucleic acid is fixed on the support, the support is contacted with a suitably labeled probe nucleic acid for about two to forty eight hours. After the above time period, the solid support is washed several times at a controlled temperature to remove unhybridized probe. The support is then dried and the hybridized material is detected by autoradiography or by spectrometric methods. When very low concentrations must be detected, the above method is slow and labor intensive, and nonisotopic labels that are less readily detected than radiolabels are frequently not suitable. The above time period may be shortened by employing techniques such as electrophoresis, which allows detection of specific nucleic acid sequences in a relatively shorter time of about 10 minutes to one hour.
A method for the enzymatic amplification of specific segments of DNA known as the polymerase chain reaction (PCR) method has been described. This in vitro amplification procedure is based on repeated cycles of denaturation, oligonucleotide primer annealing, and primer extension by thermophilic polymerase, resulting in the exponential increase in copies of the region flanked by the primers. The PCR primers, which anneal to opposite strands of the DNA, are positioned so that the polymerase catalyzed extension product of one primer can serve as a template strand for the other, leading to the accumulation of a discrete fragment whose length is defined by the distance between the 5' ends of the oligonucleotide primers.
Other methods for amplifying nucleic acids have also been developed. These methods include single primer amplification, ligase chain reaction (LCR), transcription-mediated amplification methods including 3SR and NASBA, the Q-beta-replicase method, the rolling circle amplification, and so forth. Regardless of the amplification used, the amplified product must be detected.
One method for detecting nucleic acids is to employ nucleic acid probes that have sequences complementary to sequences in the target nucleic acid. A nucleic acid probe may be, or may be capable of being, labeled with a reporter group or may be, or may be capable of becoming, bound to a support. Detection of signal depends upon the nature of the label or reporter group. Usually, the probe is comprised of natural nucleotides such as ribonucleotides and deoxyribonucleotides and their derivatives although unnatural nucleotide mimetics such as 2'-modified nucleosides, peptide nucleic acids and oligomeric nucleoside phosphonates are also used. Commonly, binding of the probes to the target is detected by means of a label incorporated into the probe. Alternatively, the probe may be unlabeled and the target nucleic acid labeled. Binding can be detected by separating the bound probe or target from the free probe or target and detecting the label. In one approach, a sandwich is formed comprised of one probe, which may be labeled, the target and a probe that is or can become bound to a surface. Alternatively, binding can be detected by a change in the signal-producing properties of the label upon binding, such as a change in the emission efficiency of a fluorescent or chemiluminescent label. This permits detection to be carried out without a separation step. Finally, binding can be detected by labeling the target, allowing the target to hybridize to a surface-bound probe, washing away the unbound target and detecting the labeled target that remains.
Direct detection of labeled target hybridized to surface-bound probes is particularly advantageous if the surface contains a mosaic of different probes that are individually localized to discrete, known areas of the surface. Such ordered arrays containing a large number of oligonucleotide probes have been developed as tools for high throughput analyses of genotype and gene expression. Oligonucleotides synthesized on a solid support recognize uniquely complementary nucleic acids by hybridization, and arrays can be designed to define specific target sequences, analyze gene expression patterns or identify specific allelic variations.
In one approach, cell matter is lysed, to release its DNA as fragments, which are then separated out by electrophoresis or other means, and then tagged with a fluorescent or other label. The resulting DNA mix is exposed to an array of oligonucleotide probes, whereupon selective attachment to matching probe sites takes place. The array is then washed and imaged so as to reveal for analysis and interpretation the sites where attachment occurred.
The goal of array fabrication is to produce a matrix of the order of 10,000 probe sites or more in an area several to tens of millimeters on a side. Each oligonucleotide probe has a length typically in the 10 to 40 base pair length. Many methods have been put forth for fabricating such arrays. In one approach the oligonucleotide probes are spotted on a suitable surface to produce an array. For this purpose, pre-synthesized probes are employed. In another approach a substrate is prepared upon which is located microdrop-sized loci at which chemical compounds are synthesized or diagnostic tests are conducted. The loci are formed by applying microdrops from which a microdrop is pulse-fed onto the surface of the substrate.
In another approach, arrays are fabricated in situ, adding one base pair at a time to a primer site. Affymetrix, for example, uses photolithography to uncover sites, which are then exposed and reacted with one of the four base pair phosphoramidites. In photolithography the surface is first coated with a light-sensitive resist, exposed through a mask and the pattern is revealed by dissolving away the exposed or the unexposed resist and, subsequently, a surface layer. A separate mask must be made for each pattern, which may involve four patterns for each base pair in the length of the probe. Much overhead is involved in preparing the masks for photolithography, which may number 80 for probes of length 20, thus rendering this technique best suited for very high volume production. There are also problems in controlling the etching reaction and in registering masks between each step.
Another in situ method employs inkjet printing technology to dispense the appropriate phosphoramidite onto the individual probe sites. For example, see U.S. Pat. No. 5,700,637 and PCT WO 95/25116.
Another method involves electrochemically patterning a surface. An electrolyte overlying the surface and an array of electrodes adjacent to the surface and in contact with the electrolyte is provided. The potential of one or more electrodes of the array is altered so as to deposit or remove or chemically modify a substance on the surface adjacent the electrode. Several such treatments may be performed in sequence using different electrodes of the array. The method may be used for step-wise chemical synthesis of, for example, oligonucleotides tethered to the surface.
In a similar approach a self-addressable, self-assembling microelectronic device is used to carry out and control multi-step and multiplex molecular biological reactions, such as biopolymer synthesis, nucleic acid hybridization, antibody-antigen reaction, and diagnostics, in microscopic formats. The device electronically can control the transport and attachment of specific binding entities and other reactants to specific microlocations.
Array plates have been discussed where a glass support surface is coated with a positive or negative photoresist substance and then exposed to light and developed to create a patterned region of a first exposed surface and a photoresist coated surface on the support. The first exposed surface is reacted with a fluoroalkylsilane to form a stable fluoroalkylsiloxane hydrophobic matrix on the first exposed surface. The photoresist coat on the surface is removed so as to form a second exposed surface, which is reacted with a hydroxy- or aminoalkylsilane so as to convert the second exposed surface to a derivatized hydrophilic binding site region and thus form the array plate.
In another approach a biological electrode array is used. Each electrode in the array is coupled to a respective sample-and-hold circuit. The electrodes and sample-and-hold circuits are integral and form an array within a single semiconductor chip, such that each sample-and-hold circuit may be loaded with a predefined voltage provided by a single, time-shared digital-to-analog converter. All of the sample-and-hold may be accessed through a multiplexer that may scan through some or all of the electrode locations. Each sample-and-hold circuit may comprise a capacitor and one or more transistor switches, which, when closed, provide electrical communication between the capacitor and a source line formed in the matrix.
The known techniques are not without limitations, however. The photolithographic approach is time consuming, and thus expensive and, due to its chemical complexity, starts to introduce errors as the probe lengths grow much beyond ten base pairs. While the inkjet method offers better probe fidelity, its spot nature also slows down fabrication, and, thus, it is best-suited for lower volume array fabrication. Also, there is a potential problem with the chemical compatibility of reactive nucleotide intermediates with inkjet printing technology and the reproducibility of spot to spot registration.
The above-mentioned biological electrode array may be limited because of the requirements of the IC fabrication required to prepare the chips. According to known IC procedures, the chips must be electrically powered during the fabrication process resulting in potential generation of noise because of the continual refresh involved. Furthermore, the chips are individually reacted chemically so as to build the desired chemical structure at each site. The drawback of this approach is the expense of chemically programming the devices either individually or in small groups.
2. Description of the Related Art
PCT application WO 98/01758 (Kovacs) discloses a multiplexed active biologic array.
U.S. Pat. No. 5,605,662 (Heller, et al.) (Heller 1)) discloses active programmable electronic devices for molecular biological analysis and diagnostics. A corresponding PCT application is WO 95/12808. The devices and systems are self-addressable, self-assembling and microelectronic.
U.S. Pat. No. 5,632,957 (Heller, et al.) (Heller 2), and corresponding PCT application WO 95/01836, discuss molecular biological diagnostic systems including electrodes. The devices and systems are self-addressable, self-assembling and microelectronic.
Heller, et al., (Heller 3) disclose apparatus and methods for active programmable matrix devices in PCT application, WO 97/12030.
Heller, et al., (Heller 4) disclose an automated molecular biological diagnostic system in PCT application, WO 96/07917.
U.S. Pat. No. 5,667,667 (Southern) discloses electrochemical treatment of surfaces.
U.S. Pat. No. 5,180,480 (Manz) discusses an apparatus for the preparation of samples especially for analytical purposes.
Austin, et al., (Austin, U.S. Pat. No. 5,427,663) disclose microlithographic array for macromolecule and cell fractionation.
U.S. Pat. No. 5,582,701 (Geis, et al.) discusses an ionic liquid-channel charge-coupled device.
Hollis, et al., (Hollis, U.S. Pat. No. 5,653,939) disclose optical and electrical methods and apparatus for molecule detection.
U.S. Pat. No. 4,832,759 (Curtis, et al.) discloses the location of biological cells in a predetermined spatial disposition relative to each other on a solid non-biological substrate.
An electrode configuration for matrix addressing of a molecular detection device is discussed by Ackley in U.S. Pat. No. 5,728,532.
PCT WO 95/25116 (Baldeschwieler, et al.) discloses a method and apparatus for performing multiple sequential reactions on a matrix.
U.S. Pat. No. 5,474,796 (Brennan) discloses a method for making array plates.
U.S. Pat. No. 5,445,934 (Fodor, et al.) discusses an array of oligonucleotides on a solid substrate.